NEB EXPRESSIONS

A scientific update from New England Biolabs Winter Edition 2012

Gibson Assembly – Building a Toolset page 3 Gibson Assembly Master Mix page 6 RE-Mix Master Mixes page 8 New NEBNext Products for NGS Library Preparation pages 10–11 Table of Contents A Letter from NEB Dear Researcher,

Feature Article Innovation is more than just change for change’s sake . Innovation is the intelligent Gibson Assembly™ – Building a modification of existing processes for improved outcomes . NEB has been developing Synthetic Biology Toolset . . . page 3 innovative tools for research for nearly 40 years, and continues to be a leader in the research, discovery and development of life sciences reagents . We take our New Products commitment to the advancement of science seriously, and we strive to offer cutting edge Gibson Assembly™ Master Mix page 6 resources to take your research to the next level . To that end, we have joined with Synthetic Phusion® Hot Start Flex to offer the Gibson Assembly Master Mix to facilitate assembly of DNA fragments DNA Polymerase ...... page 7 in your lab . This one-step, isothermic approach to in vitro recombination allows for the joining of multiple fragments with overlapping ends into a single recombinant molecule . Check out ™ RE-Mix Restriction Enzyme the feature article in this edition of NEB expressions to learn more about synthetic biology and Master Mixes ...... page 8 the innovative Gibson Assembly method of fragment assembly . HF Restriction Enzymes ...... page 9 This edition of NEB expressions also highlights our new product line of RE-Mix Master ® NEBNext Products for the Illumina Mixes, offering true one-step simplicity in your enzyme digestions . The RE-Mix line of Sequencing Platform . . . pages 10–11 Master Mixes offers speed and NEBNext® Fast DNA Library Prep Sets simplicity, but also offers the for Ion Torrent™ ...... page 11 flexibility to digest your DNA for 5 minutes or overnight — FAQs with no loss of product . Using RE-Mix Master Mixes . . page 9 Wishing you continued success in your research, Upcoming New England Biolabs Tradeshows

Visit the NEB booth at Pine branches covered with snow on the New England Biolabs’ campus. the following meetings: Photographed by Wendy Geary. • ABRF (Association for Biomolecular Resource Facilities) Orlando, FL March 17–20 Mix it up . http://conf .abrf .org/ Booth 205 RE-Mix Restriction Enzyme • AACR (American Association for Master Mixes Cancer Research) Chicago, IL March 31–April 4 Restriction Enzyme digests are now even easier! http://www .aacr .org/default .aspx With RE-Mix Master Mixes take advantage of: Booth 727 • Simplified and shortened protocols • Experimental Biology San Diego, CA • Fast digestion in 15 minutes April 21–24 (Time-Saver qualified) http://www .experimentalbiology .org • High product quality with reproducible results Booth 1210 Just add your DNA and mix!

See page 8 for more information .

2 Feature Article www.neb.com

Gibson Assembly™ – Building a Synthetic Biology Toolset In the quest to create the first bacterial cell controlled by a synthetic genome, the J. Craig Venter Institute (JCVI), with support from Synthetic Genomics, Inc. (SGI), developed a variety of powerful new DNA synthesis and assembly methodologies (1–5) to manipulate large, complex DNAs. These methods include a simple, one-step isothermal in vitro recombination technology capable of joining DNAs ranging from relatively short oligonucleotides to fragments hundreds of kilobases in length. This approach, commonly referred to as “Gibson Assembly,” is now being used in laboratories around the world to construct DNA fragments. It has the potential to improve upon traditional cloning methods and opens up a range of innovative and ultimately very useful real-world applications.

Daniel G . Gibson, Ph D. ,. Synthetic Genomics, Inc . and two-step thermocycle-based in vitro recombina- Gibson Assembly has become the most commonly Salvatore Russello, Ph D. ,. New England Biolabs, Inc . tion method utilizing these enzymes was used used of the in vitro assembly methods discussed Introduction: to join 101 overlapping DNA cassettes into four above, as it is easy-to-use, flexible and needs parts of the M. genitalium genome, each between little or no optimization, even for large, complex The use of recombinant DNA technology began 136 kb and 166 kb in size . This milestone assemblies . All that is required is input DNA with soon after the discovery of DNA ligase and marked the first assembly of a genome derived appropriate overlaps, and an appropriate mix of restriction endonucleases . Soon after, advent of from a free-living organism . At 582,970 bp, this the three enzymes – the Gibson Assembly Master the polymerase chain reaction (PCR) opened synthetic genome was the largest chemically de- Mix . DNA fragments are added to the master mix up new possibilities for amplification of specific fined DNA structure synthesized in a laboratory, and incubated at 50°C for 1 hour; the result- DNA sequences from a complex mixture of and was 18 times larger than any DNA that had ing assembly product is a fully sealed dsDNA genomic DNA . These technologies have been previously been synthesized (4) . suitable for a range of downstream applications a mainstay in the modern scientific laboratory (Figure 1) . JCVI has used Gibson Assembly to for several decades and remain useful methods Since then, two additional in vitro recombination methods have been developed by JCVI to join rapidly synthesize the entire 16,520 bp mouse for cloning potentially valuable or interesting mitochondrial genome from 600 overlapping DNA today . However, as scientists seek to work and clone DNA molecules larger than 300 kb in a single step (2-4) . The simplest of these methods 60-base oligonucleotides (3) . It was also used in with larger DNA fragments, conduct extensive combination with yeast assembly to synthesize re-engineering of genetic elements, synthesize is Gibson Assembly, a one-step isothermal ap- proach that utilizes the same three enzymatic the 1 .1 Mbp Mycoplasma mycoides genome, which whole genomes and move towards automated ap- was then activated in a recipient cell to produce activities described previously . This method can proaches, the technologies required to manipulate the first synthetic cell (1) . DNA also need to evolve . be used to join both ssDNA and dsDNAs . Investigators at the J . Craig Venter Institute (JCVI) have developed a number of in vitro Figure 1 . Overview of Gibson Assembly . enzymatic strategies to assemble short oligonu- cleotides into larger double-stranded DNA con- dsDNA fragments with overlapping ends. structs (1-4) . In 2003, JCVI made a significant A advancement in the production of a synthetic 3´ 5´ B genome by assembling the 5,386 bp genome 5´ of phiX174, a virus that infects bacteria, in just 3´ Gibson Assembly 14 days (5) . This approach involved joining 3´ 5´ synthetic oligonucleotides by polymerase cycling Add fragments to 5´ 3´ assembly, and subsequently amplifying them by Gibson Assembly 5´ Exonuclease chews back 5´ ends. PCR (5-6) . The unprecedented speed with which Master Mix. 3´ 5´ this was completed laid the foundation for con- 5´ structing larger and more complex genomes . 3´ DNA fragments anneal. In 2004, JCVI began synthesizing the Mycoplas- 3´ 5´ ma genitalium genome . It was found that overlap- 5´ 3´ ping DNA molecules could be efficiently joined DNA polymerase extends 3´ ends. using three enzyme specificities: (i) exonuclease Incubate at 50°C activity, that chews back the ends of DNA frag- for 1 hour. DNA ligase seals nicks. ments and exposes ssDNA overhangs that can anneal to their ssDNA complement; (ii) DNA polymerase activity, that fills gaps in the annealed products, and (iii) DNA ligase activity, that cova- A + B lently seals the resulting nicks in the assembly . A Fully Assembled DNA

3 Applications of Gibson Assembly: synthesizing long stretches of oligonucleotides synthetic cassettes and the desired M. mycoides ge- Cloning. Gibson Assembly eliminates the need to is the introduction of errors (9) . To ensure that nome sequence . The sequences of 16 cassettes were engineer restriction enzyme cut sites within DNA error-free molecules are obtained at a reasonable successfully edited using this approach (1) . efficiency, a strategy employed by SGI and JCVI when assembling fragments together . DNA mol- Combinatorial synthesis of DNA Fragments. involves the assembly of only eight to twelve ecules are designed such that neighboring frag- In the near future, chromosomes will be designed 60-base oligonucleotides (with 30 bp overlaps) ments contain a 20-40 bp overlapping sequence . If and synthesized for processes ranging from biofuel the DNA fragments originate from PCR products, at one time . The resulting dsDNA molecules are production to pharmaceutical manufacture . Bacteria the overlapping sequence is introduced at the sequence-verified and assembled into larger DNA and plants often carry out syntheses that far exceed 5′ ends of the primers used in the amplification fragments using the same approach . Because what can be readily achieved by the best organic reaction (Figure 2) . DNA fragments can also be as- assembly itself does not generally introduce new chemists . The genes that control desirable pathways errors, the final assembled product can be retrieved sembled with restriction enzyme digested or PCR can be chemically synthesized, placed in artificial amplified vector to form circular products suitable at high efficiencies . Using this approach, many of chromosomes, and “installed” in suitable host cells, for cloning, or for use in downstream applications, the costly and time consuming steps currently used including bacteria, yeast or plant cells . These multi- such as rolling circle amplification (RCA) . To to synthesize DNA, including PCR and an error gene pathways can be constructed in a combinato- produce these vectors by PCR, each primer needs correction, are eliminated . rial fashion, such that each member of the library to include an overlap with one end of the vector, a Site-directed mutagenesis. Gibson Assembly has a different combination of gene variants . Using restriction site (e .g ., Not I) not present within the can also be used to make rapid changes to DNA screening and selection methods, cells bearing the insert or inserts to enable it to be released from the fragments, including substitutions, deletions and pathway with the desirable trait (highest yield of vector, and an overlap with the ends of the DNA insertions . To use Gibson Assembly for mutagen- a compound, for instance) can be obtained . The fragment assembly or insert . JCVI has been using esis, the desired changes are introduced into the engineered host organism then becomes a biologic this approach to combine DNA fragments with PCR primers, within the overlapping sequences at factory used to manufacture the product specified vectors, which are then transformed into E. coli . assembly points (Figure 3) . To modify a DNA se- by the synthetic pathway . Gibson Assembly has One or more fragments have been routinely assem- quence in this way, two PCR primers are required: the potential to be used to produce combinatorial bled with general cloning vectors, such as pUC19, the first contains the desired nucleotide changes, libraries of synthetic or semisynthetic chromosomes and assembled into NEB’s pTYB1 expression vec- and the second contains the reverse complement of carrying thousands of genes . Figure 4 demon- tor (NEB #N6701) . The latter approach was used the first primer at the overlapping region . Follow- strates the combinatorial assembly of cassettes to express several methylase genes, which aided ing amplification and assembly of the fragments, produced from 60-mer oligonucleotides . Here, the genome transplantation efforts at JCVI (8) . the designed changes are incorporated into the 1,024 (210) variants of a 1 kb gene, containing 10 Assembly of large DNA constructs. Laboratories final product . The number of changes that can be single nucleotide changes, are produced from 30 worldwide are beginning to explore the use of made at once depends on the number of fragments sequence-verified cassettes . simultaneously assembled . For example, an eight- synthetic biology approaches in the production of Moving Forward pharmaceuticals, industrial compounds, antibiot- piece assembly, which contains eight assembly Synthetic & Minimal Cells. For the past 17 ics, cosmetics and alternative energy sources (7) . points, provides eight opportunities to introduce years, the genomes of many organisms have been This often requires the assembly of a genetic changes in the DNA sequence . Because the meth- sequenced and deposited in databases . It has re- pathway consisting of multiple enzymes and their od can be used to assemble large DNA fragments, cently been shown that it is possible to reverse this associated regulatory elements . Although template mutations can rapidly be made to very large pieces process and synthesize bacterial cells from digitized DNA is still required, Gibson Assembly simpli- of DNA . For example, eight modifications can be information (1) . In order to realize this vision, fies construction of these types of molecules from introduced into an 80 kb DNA molecule follow- researchers at JCVI needed tools and technologies component fragments . A long stretch of desirable ing the assembly of eight 10 kb PCR fragments . to sequence, synthesize and transplant genomes . DNA sequence (e g. ., a 40 kb genetic pathway) can This site-directed mutagenesis strategy was used Although many hurdles needed to be overcome, be broken down into several overlapping PCR during synthesis of the M. mycoides genome . The synthetic cells can now be produced in the labora- products (e .g ,. eight, 5 kb pieces), which can then cassettes comprising the synthetic genome were tory . As proof of concept, the 1 .08 Mbp M. mycoides be amplified by conventional PCR and combined ordered based on an imperfect draft sequence, JCVI-syn1 0. genome was designed, synthesized using Gibson Assembly . This approach has been which resulted in small differences between the used to move genetic pathways from one organism to another and to rapidly swap genes, promoters, terminators and ribosome binding sites . DNAs Figure 2 . PCR-Generated Vector and Insert Assembly . up to ~1 Mbp have been assembled in vitro using Gibson Assembly (2) . Forward Primer to Amplify Gene of Interest Assembly of chemically-synthesized oligonucle- Overlap Region (≥16 nt) Gene-specific Sequence (18-25 nt) Forward primer to amplify vector otides into dsDNA fragments. Gibson Assembly 5´-NNNNNNNNNNNNNNNNNN-3´ 5´-NNNNNNNNNNNNNNNNNN-3´ 3´-NNNNNNNNNNNNNNNNNN-5´ Gene of Interest 3´-NNNNNNNNNNNNNNNNNN-5´ can also be used to directly assemble oligonucle- otides into a cloning vector, such as pUC19 (3) . Reverse Primer to Amplify Vector Gene-specific Sequence (18-25 nt) Overlap Region (≥16 nt) A common problem observed when chemically Reverse Primer to Amplify Gene of Interest

4 www.neb.com

Figure 3 . Introducing changes to a desired DNA using Gibson Assembly . biology forward . As the power of DNA sequenc- ing increases and sequencing costs decrease, DNA 1F databases will continue to fill with novel genes and pathways waiting to be identified, optimized 2F 5F 1R 2R 5R pUC19 pUC19 and expressed in a heterologous host organism . 3R 4R 3F It is time to better understand how to turn these 4F DNA sequences into useful applications . PCR Deletion Insertion The ability to quickly construct whole genes and 1 2 3 4 5 1 1 2 5 Transformation genomes has the potential to accelerate research 3 4 in a variety of other fields . This capability may 2 3 4 5 also make it possible to quickly respond to Changed plasmid emerging threats, and may allow researchers to understand how “life” works . The power of large In the example above, five changes were introduced into a DNA plasmid using Gibson Assembly. Each primer contains a change introduced by PCR. The overlapping PCR fragments with each change at the assembly point are then combined using Gibson Assembly. scale DNA synthesis will dramatically impact (credit: Mikkel Algire, JCVI). the way research is done and vastly acceler- ate the pace of science . The Gibson Assembly Master Mix provides a new and powerful tool for and assembled, starting from the digitized genome Conclusion biotechnology, whose most far-reaching benefits may not yet even be envisioned . sequence, and transplanted into a Mycoplasma Gibson Assembly is a simple and robust method capricolum recipient cell to create new M. mycoides that enables the simultaneous production of many References cells controlled only by the synthetic chromo- different combinations of genes and pathways, 1 . Gibson, D .G . et al . (2010) Science, 239, 52–56 . 2 . Gibson, D .G . et al . (2009) Nature Methods, 6, 343–345 . some . The only DNA present in the cells is the accelerating the progress of synthetic biology . designed synthetic DNA, including “watermark” 3 . Gibson, D .G . et al . (2010) Nature Methods, 7, 901–903 . Furthermore, this powerful technology has the po- 4 . Gibson, D .G . et al . (2008) Science, 319, 1215–1220 . sequences, and other designed gene deletions and tential to help turn DNA sequence into genes and 5 . Smith et al . (2003) PNAS, 100, 15440–15445 . polymorphisms and mutations acquired during the pathways useful in the production of biofuels, in- 6 . Stemmer, W .P . et al . (1995) Gene, 164, 49–53 . building process . The new cells have the expected dustrial compounds, pharmaceuticals and vaccines . 7 . Endy, D . (2005) Nature, 438, 449–453 . phenotypic properties and are capable of continu- The synthesis of genes and pathways, and even 8 . Lartique, C . et al . (2009) Science, 325, 1693–1696 . 9 . Carr et al . (2004) Nucleic Acids Res . 32, e162 . ous self-replication (1) . The M. mycoides genome small genomes, has been made easier with Gibson ™ is currently the largest chemically defined DNA Assembly, helping to move the field of synthetic Gibson Assembly is a trademark of Synthetic Genomics, Inc. structure that has been synthesized in a laboratory . It is almost twice as large as the synthetic M. geni- talium genome reported in 2008, and more than an Figure 4 . Combinatorial gene synthesis . order of magnitude larger than any reported DNA sequence synthesized outside JCVI . What has been learned in this “proof of concept” experiment can now be applied to designing and producing new 23 = 8 21 = 2 21 = 2 organisms with useful properties . Further, researchers at JCVI have already begun working on their ultimate objective: to synthesize a minimal cell with only the machinery necessary for independent life . Now that a living cell can be 21 = 2 24 = 16 combinations produced from a synthetic genome, components of a synthetic genome can be removed and trans- planted in an iterative fashion until only the essen- tial genes are present and the genome is as small 40bp overlap 60-mer Variant 60-mer Variant nucleotide as possible . This will help to better understand the between cassettes function of every gene in a cell and what DNA is required to sustain life in its simplest form . Gibson Ten overlapping 60-mer oligonucleotides are recombined into five cassettes (light blue rectangles). Following sequence verification of these Assembly is one of the core technologies that will cassettes, they are recombined (indicated by the X) into the full-length gene in a second stage. Recombination of the cassettes is possible because 40 bp overlaps have been added to the cassettes. Within each cassette, there are between 1 and 4 positions in which 2 nucleotides are acceptable. be used to achieve these goals . At these locations, a variant 60-mer oligonucleotide can also be assembled. This equates to 2-16 different combinations for each cassette. In order to produce every different combination (23 x 21x 21 x 24 x 21 = 1,024) of the full-length gene, every different cassette can be pooled and assembled into a cloning vector to form a circular DNA, cloned into E. coli, and then sequenced.

5 New Products

™ Gibson Assembly Master Mix Advantages Gibson Assembly was developed by Dr. Daniel Gibson and his colleagues at the J. Craig Venter Institute and • Increased number of successful assem- licensed to NEB by Synthetic Genomics, Inc. It allows for the successful assembly of multiple DNA fragments, bly products, particularly for longer regardless of fragment length or end compatibility. It has been rapidly adopted by the synthetic biology community or greater number of fragments due to its ease-of-use, flexibility and suitability for large DNA constructs. • Flexible sequence design with no need to engineer cloning sites (scar- Gibson Assembly efficiently joins multiple overlapping DNA fragments in a single-tube isothermal less cloning) reaction (1,2) . The Gibson Assembly Master Mix includes three different enzymatic activities that perform in a single buffer (see Figure 1, page 3): • Does not require clean up of PCR products prior to assembly • The exonuclease creates a single-stranded 3´ overhang that facilitates the annealing of fragments that share complementarity at one end . • Complex assembly achieved in 1 hour • The polymerase fills in gaps within each annealed fragment. • DNA can be used immediately for transformation, or as template for • The DNA ligase seals nicks in the assembled DNA. PCR or RCA The end result is a double-stranded, fully-sealed DNA molecule that can serve as template for PCR, • Easily adapted for multiple DNA RCA or a variety of other molecular biology applications, including direct transformation . The meth- manipulations, including site-directed od has been successfully used by Gibson’s group and others to assemble oligonucleotides, DNA with mutagenesis, insertions and deletions varied overlaps (15–80 bp) and fragments hundreds of kilobases long (1–3) . In contrast to other methods, Gibson Assembly is suitable for a wide range of different types of 1 .Gibson, D .G . et al . (2009) Nature Methods, 343–345 . assemblies, including large numbers of DNA fragments . The figure below shows that Gibson 2 .Gibson, D .G . et al . (2010) Nature Methods, 901–903 . Assembly outperforms other methods for assembly of six fragments . 3 .Gibson, D .G . Personal communication .

Assembly of DNA fragments using methods from three different suppliers .

120

100

80 2 Piece Assembly (3.0 & 5.4 kb 60 with 15–20 bp overlap) Looking for Synthetic 6 Piece Assembly 40 (five 0.4 kb inserts Gene Fragments?

% Correct Assembly and a 2.7 kb vector 20 with 40 bp overlap) Integrated DNA Technologies (IDT) offers a range of synthetic gene products 0 ™ NEB Supplier A Supplier B manufactured using Ultramer oligonucle- otides, the highest fidelity next generation The assemblies were conducted in accordance with the manufacturer’s recommended synthesis technology available . gBlocks™ protocols. Following assembly and transformation, a portion of the resulting colonies Gene Fragments are linear, high-fidelity, were tested for the presence of a PCR fragment corresponding to a correct, fully assembled construct, by colony PCR, using primers that amplify the entire fragment. double-stranded genomic blocks up to 500 While all three vendors’ methods perform a 2 fragment assembly well, the Gibson As- bp in length, and are ideal for use with the sembly Master Mix outperforms other suppliers for larger numbers of fragments. Gibson Assembly method . Integrated DNA Technologies also offers complete custom Ordering Information synthetic genes, delivered in a vector ready for use in a variety of applications . For PRODUCT NEB # SIZE more information, visit www idtdna. com. . Gibson Assembly Master Mix E2611S/L 10/50 reactions

Gibson™ is a trademark of Synthetic Genomics, Inc. gBlocks™ and Ultramer™ are trademarks of Integrated DNA Technologies, Inc. Manufactured by New England Biolabs, Inc. under license from Synthetic Genomics, Inc.

6 www.neb.com

® Phusion Hot Start Flex Advantages DNA Polymerase • Robust Reactions – maximum success with minimal optimization Phusion Hot Start Flex DNA Polymerase delivers additional flexibility to reaction setup and cycling • Extreme Fidelity – highest of any conditions . In contrast to chemically modified or antibody-based hot start polymerases, Phusion available thermostable polymerase Hot Start Flex utilizes an innovative synthetic aptamer . This structure binds to the polymerase (50X greater than Taq) through non-covalent interactions, blocking activity during reaction setup . The polymerase is activated during normal cycling conditions, allowing reactions to be set up at room temperature . • High Speed – dramatically reduced Phusion Hot Start Flex does not require a separate high temperature activation step, shortening extension times reaction times and increasing ease-of-use . Phusion Hot Start Flex is an ideal choice for high speci- • High Yield – increased product yield ficity amplification over a flexible range of annealing temperatures and offers robust amplification using minimal amount of enzyme on a wide variety of templates . • Versatile – can be used for routine PCR, as well as long or difficult templates Phusion Hot Start Flex DNA Polymerase delivers robust amplification for a • Hot Start – flexible formulation allows wide range of templates . room temperature reaction setup and no activation step

1.3 kb 2.3 kb 3.9 kb

P C A P C A P C A bp M M M 4,900 2,900 1,900 1,100 P = Phusion Hot Start Flex (Aptamer-based) 700 C = Chemical-based 500 Hot Start Polymerase A = Antibody-based Hot Start Polymerase 300

All amplicons are from human Jurkat template except for the 1.3 kb C. elegans amplicon. Amplicon sizes are indicated above the gel. All reactions were conducted in duplicate according to manufacturers’ instructions using 30 cycles and visualized via microfluidic LabChip® analysis.

Ordering Information PRODUCT NEB # SIZE Phusion® Hot Start Flex DNA Polymerase M0535S/L 100/500 units Phusion® Hot Start Flex 2X Master Mix M0536S/L 100/500 rxns (50 µl vol) Phusion® High-Fidelity DNA Polymerase M0530S/L 100/500 units Phusion® High-Fidelity PCR Master Mix with HF Buffer M0531S/L 100/500 rxns (50 µl vol) Phusion® High-Fidelity PCR Master Mix with GC Buffer M0532S/L 100/500 rxns (50 µl vol) Phusion® PCR Kit E0553S/L 50/200 rxns (50 µl vol) COMPANION PRODUCTS Phusion® HF Buffer Pack B0518S 6 .0 ml Phusion® GC Buffer Pack B0519S 6 .0 ml Detergent-free Phusion® HF Buffer Pack B0520S 6 .0 ml Detergent-free Phusion® GC Buffer Pack B0521S 6 .0 ml

Phusion DNA Polymerase was developed by Finnzymes Oy, now a part of Thermo Fisher Scientific. Phusion DNA Polymerase is now manufactured by New England Biolabs, Inc. under agreement with, and under the performance specifications of Thermo Fisher Scientific. Phusion® is a registered trademark and property of Thermo Fisher Scientific. For licensing information, visit www.neb.com.

7 New Products

™ RE-Mix Restriction Enzyme Advantages Master Mixes • Convenience – easy-to-use master mix format contains enzyme, buffer, BSA For over 35 years, New England Biolabs has been developing innovative solutions for molecular and loading dye biology applications . A respected leader in the field of restriction enzyme biology, NEB is pleased to • Simple – just add DNA and water offer the first selection of restriction enzyme master mixes that bring total simplicity and convenience to restriction enzyme digests . RE-Mix Restriction Enzyme Master Mixes contain enzyme, buffer, BSA • Time-Saver qualified – digests sub- and loading dye; all that is required is the addition of DNA and water . All RE-Mixes are Time-Saver strate DNA in 15 minutes qualified and will digest DNA in 15 minutes . Unlike other “fast” restriction enzymes, they can also • Flexibility – obtain the same results be used in overnight reactions without any unwanted cleavage . With easy-to-follow protocols and whether you choose to digest for flexible reaction conditions, RE-Mix enzymes from NEB offer maximum convenience . 15 minutes or overnight • Low vial price Simple & fast – Just add DNA! L 5 min 15 min 1 hr o/n RE-Mix DNA Master Mix

Incubate and load directly onto gel

pXba DNA was digested with EcoRV-HF™ RE-Mix™ according to the recommended protocol. Lane L is the TriDye™ 2-Log DNA Ladder (NEB #N3270). The same results are obtained whether incubated for 5–15 minutes, 1 hour or overnight.

FAQ Spotlight – RE-Mix Master Mixes Q: Can I use RE-Mix Mster Mixes in a double digest? A: Yes, RE-Mix Master Mixes can be used together in a double digest reaction. For this, we recommend setting up a 40 µl reaction containing 2 µl of each RE-Mix Master Mix.

Q: If the restriction enzyme that I am using requires BSA, do I need to add it to the reaction when using RE-Mix? A: RE-Mix Master Mixes already contain enzyme, buffer, BSA and loading dye. No additional BSA is needed.

Q: Do I need to stop the reaction or add loading dye prior to agarose gel electrophoresis? A: RE-Mix Master Mixes already contain a loading dye and can be loaded directly onto an agarose gel.

Visit www.NEBREMIX.com for more information or to download a datacard 8 www.neb.com

Ordering Information PRODUCT NEB # # OF REACTIONS Introducing the new AgeI-HF™ RE-Mix™ R5552S 25 restriction enzyme AscI RE-Mix™ R5558S 50 challenge from NEB BstEII-HF™ RE-Mix™ R5162S 100

™ ™ EcoRI-HF RE-Mix R5101S 500 C EcoRV-HF™ RE-Mix™ R5195S 200 GTAGCT A KpnI-HF™ RE-Mix™ R5142S 200 MfeI-HF™ RE-Mix™ R5589S 25 T GA G NcoI-HF™ RE-Mix™ R5193S 50 AGT C ™ ™ NheI-HF RE-Mix R5131S 50 G NotI-HF™ RE-Mix™ R5189S 25 CT T ™ A C PacI RE-Mix R5547S 25 GATACG SalI-HF™ RE-Mix™ R5138S 100 ScaI-HF™ RE-Mix™ R5122S 50 Visit SpeI RE-Mix™ R5133S 50 www.NEBcutitout.com XbaI RE-Mix™ R5145S 150 XhoI RE-Mix™ R5146S 200 to test your skills today

RE-Mix™ is a trademark of New England Biolabs, Inc.

High-Fidelity (HF) Restriction Enzymes Benefits of HF Gone are the days of buffer compatibility analysis and sub-optimal enzyme conditions! HF restric- Restriction Enzymes: tion enzymes are all 100% active in a single buffer: NEBuffer 4 . This simplifies double digest reactions and eliminates the need for setting up sequential digests . HF enzymes share the same site Engineered for performance under a specificity and cleavage efficiency as their wild-type counterparts, but offer the increased flexibility wide range of conditions and convenience that you need in your laboratory . Whether you have 5 minutes or want to leave Reduced star activity eliminates your reaction overnight, restriction enzyme digests with HF enzymes produce the same, reliable unwanted cleavage results . Set up your digests with reaction-specific enzyme concentrations, without fear of risking One buffer convenience with no loss product integrity to star activity . HF enzymes offer added flexibility and functionality without of performance added cost – they’re available for the same price-per-unit as the standard version . Time-Saver qualified for 5-15 minute Three new HF’s have been added to our growing list: digests PRODUCT NEB # SIZE Added flexibility without added cost BmtI-HF™ R3658S/L 300/1,500 units BstEII-HF™ R3162S/L 2,000/10,000 units SpeI-HF™ R3133S/L 500/2,500 units

Visit www.neb.com/HF for a complete list of HF Enzymes available .

9 Take the next step

New Products New NEBNext® Products for the Illumina Sequencing Platforms NEBNext Singleplex Oligos for Illumina® Advantages ® NEBNext Multiplex Oligos for Illumina (Index Primers 1-12) • High library yields New England Biolabs now supplies two new NEBNext modules that enable singleplex or multiplex • Efficient adaptor ligation library construction of up to 12 samples: • Module format enables flexibility in • The NEBNext Singleplex Oligos Module contains the required adapter for ligation to end-repaired, library prep workflow dA-tailed DNA, as well as primers for singleplex library construction . • Low price per reaction • The NEBNext Multiplex Oligos Module contains the required adapter for ligation and the primers • Singleplex kit is also multiplex required for library amplification by PCR . The multiplex set enables incorporation of the barcodes at compatible the PCR amplification step . Oligo modules are compatible with existing NEBNext reagent sets and modules for library preparation of DNA, ChIP-Seq and mRNA libraries for sequencing with all current Illumina instruments .

DNA Library Prep Workflow for Illumina .

Ligate PCR to enrich/ Scan this code or visit Fragmentation Repair dA-Tail adaptors amplify final of purified DNA fragment fragment onto DNA adaptor modified www.neb.com/NEBNextnews2 STEPS ends ends fragments fragmented sample to sign up for

dsDNA DNA Library Prep Reagent Set for Illumina Validation the NEBNext Fragmentase by Illumina® DNA Library Prep Master Mix Set for Illumina sequencing bimonthly Products

NEBNext NEBNext End Repair dA-Tailing Quick Ligation e-newsletter Module Module Module

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Conversion of purified mRNA to DNA followed by library preparation .

mRNA Ethanol 1st Strand 2nd Strand End dA Adaptor PCR Fragmentation Precipitation Synthesis Synthesis Repair Tailing Ligation Enrichment STEPS

mRNA Library Prep Reagent Set for Illumina Validation by Illumina® mRNA Library Prep Master Mix Set for Illumina sequencing Products NEBNext NEBNext Magnesium RNA mRNA Second Strand End Repair dA-Tailing Quick Ligation Fragmentation Module Synthesis Module Module Module Module

RNaseIII RNA Singleplex Oligos for Illumina Fragmentation Module Multiplex Oligos for Illumina

OrderingmRNA InformationEthanol 1st Strand 2nd Strand End Repair/ Adaptor Fragmentation Precipitation Synthesis Synthesis dA Tailing Ligation PRODUCTSTEPS NEB # SIZE NEBNext Singleplex Oligos for Illumina E7350S/L 12/60 rxns mRNA Library Prep Reagent Set for 454 Validation ® ™ NEBNext is a registered trademark of New England Biolabs, Inc. NEBNext Multiplex Oligos for Illumina (Index Primers 1–12) E7335S/Lby 454 24/96 rxns Illumina® is a registered trademark of Illumina, Inc. mRNA Library Prep Master Mix Set for 454 sequencing Products

10 NEBNext Magnesium RNA mRNA Second Strand Fragmentation Module Synthesis Module

RNaseIII RNA Fragmentation Module

mRNA Ethanol 1st Strand 2nd Strand End Adaptor PCR Fragmentation Precipitation Synthesis Synthesis Repair Ligation Enrichment STEPS

Validation mRNA Library Prep Set for SOLiD by SOLiD™ sequencing Magnesium RNA mRNA Second Strand Products NEBNext NEBNext Fragmentation Module Synthesis Module

RNaseIII RNA Fragmentation Module www.neb.com

NEBNext Multiplex Small RNA Library Prep Set Advantages for Illumina (1-12) • Novel protocol offers improved yields and substantially reduced adaptor- NEBNext Small RNA Library Prep Set for Illumina dimer formation (Multiplex Compatible) • Enables multiplexing These new NEBNext sets include oligos, enzymes, buffers and protocols for construction of small • Low price per reaction RNA libraries for single read and paired-end read sequencing, using all current Illumina sequencing instruments . • The NEBNext Multiplex Small RNA Library Prep Set enables multiplexing of up to 12 samples. • The NEBNext Small RNA Library Prep Set is suitable for singleplex sequencing, but is also multiplex compatible . The NEBNext Fast DNA Library Prep Sets for Ion Torrent streamline library preparation workflow

Novel NEBNext Small RNA library preparation workflow for Illumina .

Primer 1st Strand PCR Size 3´ Ligation Hybridization 5´ Ligation Synthesis Amplification Selection STEPS

Validation by Illumina® Small RNA Library Prep Set for Illumina (Multiplex Compatible) sequencing Validation Products ® NEBNext NEBNext Multiplex Small RNA Library Prep Set for Illumina (1–12) by Illumina sequencing

Ordering Information PRODUCT NEB # SIZE NEBNext Multiplex Small RNA Library Prep Set for Illumina (1–12) E7300S/L 24/96 rxns NEBNext Small RNA Library Prep Set for Illumina (Multiplex Compatible) E7330S/L 24/96 rxns

™ Now Available for the Ion Torrent Platform Advantages The NEBNext Fast DNA Library Prep Sets for Ion Torrent streamline library preparation workflow and • Fast (just over 2 hours) with minimal decrease hands-on-time . NEBNext sets are available with or without fragmentation reagents . hands-on-time

Fragment, Add • High yield Set Up Amplify STEPS End Repair, Adaptors, Ligate Clean Up, Clean Up Reaction Heat Ligase, Size Select (4–8 Cycles) • Simple protocol Inactivate Buffers • Streamlined workflow TOTAL HANDS-ON Compatible with a broad range of TIME 2 min. 0 min. 1 min. 0 min. 5 min. 1 min. 3 min. 12 min. • sample input (100 ng – 1 µg) TOTAL TIME 2 min. 30 min. 1 min. 15 min. 30 min. 35–46 min. 12 min. 125–136 min. • Optional enzyme-based fragmentation • Multiplex compatible

PRODUCT NEB # SIZE NEBNext Fast DNA Fragmentation & Library Prep Set for Ion Torrent E6285S/L 10/50 rxns NEBNext Fast DNA Library Prep Set for Ion Torrent E6270S/L 10/50 rxns

11 DNA CLONING DNA AMPLIFICATION & PCR EPIGENETICS RNA ANALYSIS SAMPLE PREP FOR NEXT GEN SEQUENCING PROTEIN EXPRESSION & ANALYSIS New England Biolabs, Inc ,. 240 County Road, Ipswich, MA 01938-2723 CELLULAR ANALYSIS

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